Assay methods

ABSTRACT

The present invention is directed to methods for reducing cross-reactivity between species employed in multiplexed immunoassays.

CROSS REFERENCE TO RELATED APPLICATIONS

Reference is made to U.S. Provisional Application Ser. No. 61/779,050,filed Mar. 13, 2013, the disclosure of which is incorporated herein byreference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in the ASCII text file, named as29901_sequencelisting.txt of 2 KB, created on Mar. 12, 2014, andsubmitted to the United States Patent and Trademark Office via EFS-Web,is incorporated herein by reference.

REFERENCE FIELD OF THE INVENTION

The present invention is directed to improved methods for conductingimmunoassays. The methods are designed to amplify signals inimmunoassays and anchor immunoassay complexes employed therein.

BACKGROUND OF THE INVENTION

A substantial body of literature has been developed concerningtechniques that employ binding reactions, e.g., antigen-antibodyreactions, nucleic acid hybridization and receptor-ligand reactions, forthe sensitive measurement of analytes of interest in samples. The highdegree of specificity in many biochemical binding systems has led tomany assay methods and systems of value in a variety of marketsincluding basic research, human and veterinary diagnostics,environmental monitoring and industrial testing. The presence of ananalyte of interest may be measured by directly measuring theparticipation of the analyte in a binding reaction. In some approaches,this participation may be indicated through the measurement of anobservable label attached to one or more of the binding materials.

While the sandwich immunoassay format provides excellent sensitivity andspecificity in many applications, some analytes are present atconcentrations that are too low for detection by conventionalimmunoassay techniques. The performance of sandwich immunoassays canalso be limited by the non-specific binding of detection antibodies andby the instability of sandwich complexes comprising high off-rateantibodies. There is a need for new techniques for improving sandwichimmunoassay performance by improving sensitivity, reducing non-specificbinding and improving the stability of sandwich complexes.

SUMMARY OF THE INVENTION

The invention provides a method of detecting an analyte of interest in asample comprising: (a) contacting the sample with a surface comprising(i) a binding reagent for the analyte, and (ii) an anchoring reagentcomprising an anchoring oligonucleotide sequence complementary to anamplicon sequence, and binding the analyte to the binding reagent toform a surface-bound complex; (b) contacting the surface-bound complexwith a first detection reagent for the analyte comprising a firstproximity probe and a second detection reagent for the analytecomprising a second proximity probe to form a detection complexcomprising said binding reagent, said analyte and said first and seconddetection reagents; (c) contacting the detection complex formed in (b)with one or more connector oligonucleotides including a first connectorprobe complementary to a first region of the first proximity probe and afirst region on the second proximity probe, and a second connector probecomplementary to a second non-overlapping region of the first proximityprobe and a second non-overlapping region of the second proximity probe,the contacting step (c) is performed under conditions sufficient toligate the first and second proximity probes to form a target sequence;(d) amplifying the target sequence to generate an amplicon comprising aplurality of detection sequences and an anchoring sequence complement;(e) hybridizing the anchoring sequence to the anchoring sequencecomplement; (f) hybridizing a plurality of detection probes to theplurality of detection probe sequences; and (g) measuring the amount ofanalyte bound to the surface.

Another embodiment is a method of detecting an analyte of interest in asample comprising: (a) contacting the sample with a surface comprising(i) a binding reagent for the analyte, and (ii) an anchoring reagentcomprising an anchoring sequence complementary to an amplicon sequence,wherein the contacting step forms a surface-bound complex between theanalyte and the binding reagent; (b) contacting the surface-boundcomplex with a first detection reagent for the analyte comprising afirst proximity probe and a second detection reagent for the analytecomprising a second proximity probe to form a detection complexcomprising said binding reagent, said analyte and said first and seconddetection reagents; (c) contacting the detection complex formed in (b)with one or more connector oligonucleotides including a firstcircularization probe complementary to a first region of the firstproximity probe and a first region on the second proximity probe, and asecond circularization probe complementary to a second non-overlappingregion of the first proximity probe and a second non-overlapping regionof the second proximity probe, the contacting step (c) is performedunder conditions sufficient to form a circular DNA template; (d)amplifying the circular DNA template to generate an amplicon comprisinga plurality of detection sequences and an anchoring sequence complement;(e) hybridizing the anchoring sequence to the anchoring sequencecomplement; (f) hybridizing a plurality of detection probes to theplurality of detection probe sequences; and (g) measuring the amount ofanalyte bound to the surface.

The method also provides a method of detecting an analyte of interest ina sample comprising: (a) contacting the sample with two detectionreagents for the analyte to form a detection complex, wherein the twodetection reagents comprise a first proximity probe and a secondproximity probe, respectively; (b) contacting the detection complexformed in (a) with a surface comprising (i) a binding reagent for theanalyte and (ii) an anchoring reagent comprising an anchoring sequencecomplementary to an amplicon sequence, wherein the contacting step (b)forms a surface-bound complex; (c) contacting the surface-bound complexformed in (b) with one or more connector oligonucleotides including afirst connector probe complementary to a first region of the firstproximity probe and a first region on the second proximity probe, and asecond connector probe complementary to a second non-overlapping regionof the first proximity probe and a second non-overlapping region of thesecond proximity probe, the contacting step (c) is performed underconditions sufficient to ligate the first and second proximity probes toform a target sequence; (d) amplifying the target sequence to generatean amplicon comprising a plurality of detection sequences and ananchoring sequence complement; (e) hybridizing the anchoring sequence tothe anchoring sequence complement; (f) hybridizing a plurality ofdetection probes to the plurality of detection probe sequences; and (g)measuring the amount of analyte bound to the surface.

Also provided is a method of detecting an analyte of interest in asample comprising: a. Contacting the sample with two detection reagentsfor the analyte to form a detection complex, wherein the two detectionreagents comprise a first proximity probe and a second proximity probe,respectively; (b) contacting the detection complex formed in (a) with asurface comprising (i) a binding reagent for the analyte and (ii) ananchoring reagent comprising an anchoring sequence complementary to anamplicon sequence, wherein the contacting step (b) forms a surface-boundcomplex; (c) contacting the surface-bound complex formed in (b) with oneor more connector oligonucleotides including a first circularizationprobe complementary to a first region of the first proximity probe and afirst region on the second proximity probe, and a second circularizationprobe complementary to a second non-overlapping region of the firstproximity probe and a second non-overlapping region of the secondproximity probe, the contacting step (c) is performed under conditionssufficient to form a circular DNA template; (d) amplifying the circularDNA template to generate an amplicon comprising a plurality of detectionsequences and an anchoring sequence complement; (e) hybridizing theanchoring sequence to the anchoring sequence complement; (f) hybridizinga plurality of detection probes to the plurality of detection probesequences; and (g) measuring the amount of analyte bound to the surface.

Also contemplated is a method of detecting a plurality of analytes ofinterest in a sample comprising: (a) contacting the sample with asurface comprising a plurality of discrete binding domains, each bindingdomain comprising (i) a binding reagents for an analyte, and (ii) ananchoring reagent comprising an anchoring sequence complementary to anamplicon sequence, wherein the contacting step forms a plurality ofsurface-bound complexes; (b) contacting each of the surface-boundcomplexes with two detection reagents for the analyte to form aplurality of detection complexes, wherein the two detection reagentscomprise a first proximity probe and a second proximity probe,respectively; (c) contacting each detection complex formed in (b) withone or more connector oligonucleotides including a first connector probecomplementary to a first region of the first proximity probe and a firstregion on the second proximity probe, and a second connector probecomplementary to a second non-overlapping region of the first proximityprobe and a second non-overlapping region of the second proximity probe,the contacting step (c) is performed under conditions sufficient toligate the first and second proximity probes to form a plurality oftarget sequences; (d) amplifying the plurality of target sequences togenerate a plurality of amplicons each comprising a detection sequenceand an anchoring sequence complement; (e) hybridizing each anchoringsequence to the anchoring sequence complement; (f) hybridizing aplurality of detection probes to the plurality of detection probesequences; and

(g) measuring the amount of analytes bound to the surface.

Moreover, the invention includes a method of detecting a plurality ofanalytes of interest in a sample comprising: (a) contacting the samplewith a surface comprising a plurality of discrete binding domains, eachbinding domain comprising (i) a binding reagent for an analyte, and (ii)an anchoring reagent comprising an anchoring sequence complementary toan amplicon sequence, wherein the contacting step forms a plurality ofsurface-bound complexes; (b) contacting each of the surface-boundcomplexes with two detection reagents for the analyte to form aplurality of detection complexes, wherein the two detection reagentscomprise a first proximity probe and a second proximity probe,respectively; (c) contacting each detection complex formed in (b) withone or more connector oligonucleotides including a first circularizationprobe complementary to a first region of the first proximity probe and afirst region on the second proximity probe, and a second circularizationprobe complementary to a second non-overlapping region of the firstproximity probe and a second non-overlapping region of the secondproximity probe, the contacting step (c) is performed under conditionssufficient to form a plurality of circular DNA templates; (d) amplifyingthe plurality of circular DNA templates to generate a plurality ofamplicons each comprising a plurality of detection sequences and ananchoring sequence complement; (e) hybridizing each anchoring sequenceto the anchoring sequence complement; (f) hybridizing a plurality ofdetection probes to the plurality of detection probe sequences; and (g)measuring the amount of analytes bound to the surface.

In addition, the invention provides a kit for the measurement of ananalyte of interest in a sample, the kit comprising: (a) a surfacecomprising a binding reagent for the analyte and an anchoring reagentcomprising an anchoring sequence complementary to an amplicon sequence;and (b) in one or more containers, compartments, or vessels: (i) twodetection reagents for the analyte, wherein the two detection reagentscomprise a first proximity probe and a second proximity probe,respectively; (ii) one or more connector oligonucleotides including afirst connector probe complementary to a first region of the firstproximity probe and a first region on the second proximity probe, and asecond connector probe complementary to a second non-overlapping regionof the first proximity probe and a second non-overlapping region of thesecond proximity probe; and (iii) one or more detection probescomplementary to the detection probe sequence.

The invention includes a kit for the measurement of an analyte ofinterest in a sample, the kit comprising: (a) a surface comprising abinding reagent for the analyte and an anchoring reagent comprising ananchoring sequence complementary to an amplicon sequence; and (b) in oneor more containers, compartments, or vessels: (i) two detection reagentsfor the analyte, wherein the two detection reagents comprise a firstproximity probe and a second proximity probe, respectively; (ii) one ormore connector oligonucleotides including a first circularization probecomplementary to a first region of the first proximity probe and a firstregion on the second proximity probe, and a second circularization probecomplementary to a second non-overlapping region of the first proximityprobe and a second non-overlapping region of the second proximity probe;and (iii) one or more detection probes complementary to the detectionprobe sequence.

Another embodiment is a kit for the measurement of a plurality ofanalytes of interest in a sample, the kit comprising: (a) a surfacecomprising a plurality of discrete binding domains, each binding domaincomprising (i) a binding reagent for an analyte and (ii) an anchoringreagent comprising an anchoring sequence complementary to an ampliconsequence; and (b) in one or more containers, compartments, or vessels:(i) two detection reagents for each analyte, wherein the two detectionreagents comprise a first proximity probe and a second proximity probe,respectively; (ii) one or more connector oligonucleotides including afirst circularization probe complementary to a first region of the firstproximity probe and a first region on the second proximity probe, and asecond circularization probe complementary to a second non-overlappingregion of the first proximity probe and a second non-overlapping regionof the second proximity probe; and (iii) one or more detection probescomplementary to the detection probe sequence.

The invention also provides a method of detecting an analyte of interestin a sample comprising: (a) contacting the sample with a surfacecomprising a binding reagent for the analyte, wherein the contactingstep forms a surface-bound complex between the analyte and the bindingreagent; (b) contacting the surface-bound complex with two detectionreagents for the analyte to form a detection complex, wherein the twodetection reagents comprise a first proximity probe and a secondproximity probe, respectively; (c) contacting the detection complexformed in (b) with one or more connector oligonucleotides including afirst connector probe complementary to a first region of the firstproximity probe and a first region on the second proximity probe, and asecond connector probe complementary to a second non-overlapping regionof the first proximity probe and a second non-overlapping region of thesecond proximity probe, the contacting step (c) is performed underconditions sufficient to ligate the first and second proximity probes toform a target sequence; (d) amplifying the target sequence to generatean amplicon comprising a plurality of detection sequences; (e)hybridizing a plurality of detection probes to the plurality ofdetection probe sequences; and (f) measuring the amount of analyte boundto the surface.

Still further, the invention includes a method of detecting an analyteof interest in a sample comprising: (a) contacting the sample with asurface comprising a binding reagent for the analyte, wherein thecontacting step forms a surface-bound complex between the analyte andthe binding reagent; (b) contacting the surface-bound complex with twodetection reagents for the analyte to form a detection complex, whereinthe two detection reagents comprise a first proximity probe and a secondproximity probe, respectively; (c) contacting the detection complexformed in (b) with one or more connector oligonucleotides including afirst circularization probe complementary to a first region of the firstproximity probe and a first region on the second proximity probe, and asecond circularization probe complementary to a second non-overlappingregion of the first proximity probe and a second non-overlapping regionof the second proximity probe, the contacting step (c) is performedunder conditions sufficient to form a circular DNA template; (d)amplifying the circular DNA template to generate an amplicon comprisinga plurality of detection sequences; (e) hybridizing a plurality ofdetection probes to the plurality of detection probe sequences; and (f)measuring the amount of analyte bound to the surface.

The invention contemplates a method of detecting an analyte of interestin a sample comprising: (a) contacting the sample with two detectionreagents for the analyte to form a detection complex, wherein the twodetection reagents comprise a first proximity probe and a secondproximity probe, respectively; (b) contacting the detection complexformed in (a) with a surface comprising a binding reagent for theanalyte, wherein the contacting step (b) forms a surface-bound complex;(c) contacting the surface-bound complex formed in (b) with one or moreconnector oligonucleotides including a first connector probecomplementary to a first region of the first proximity probe and a firstregion on the second proximity probe, and a second connector probecomplementary to a second non-overlapping region of the first proximityprobe and a second non-overlapping region of the second proximity probe,the contacting step (c) is performed under conditions sufficient toligate the first and second proximity probes to form a target sequence;(d) amplifying the target sequence to generate an amplicon comprising aplurality of detection sequences and an anchoring sequence complement;(e) hybridizing a plurality of detection probes to the plurality ofdetection probe sequences; and (f) measuring the amount of analyte boundto the surface.

Also provided is a method of detecting an analyte of interest in asample comprising: (a) contacting the sample with two detection reagentsfor the analyte to form a detection complex, wherein the two detectionreagents comprise a first proximity probe and a second proximity probe,respectively; (b) contacting the detection complex formed in (a) with asurface comprising a binding reagent for the analyte, wherein thecontacting step (b) forms a surface-bound complex; (c) contacting thesurface-bound complex formed in (b) with one or more connectoroligonucleotides including a first circularization probe complementaryto a first region of the first proximity probe and a first region on thesecond proximity probe, and a second circularization probe complementaryto a second non-overlapping region of the first proximity probe and asecond non-overlapping region of the second proximity probe, thecontacting step (c) is performed under conditions sufficient to form acircular DNA template; (d) amplifying the circular DNA template togenerate an amplicon comprising a plurality of detection sequences; (e)hybridizing a plurality of detection probes to the plurality ofdetection probe sequences; and (f) measuring the amount of analyte boundto the surface.

Further provided is a method of detecting a plurality of analytes ofinterest in a sample comprising: (a) contacting the sample with asurface comprising a plurality of discrete binding domains, each bindingdomain comprising a binding reagents for an analyte, wherein thecontacting step forms a plurality of surface-bound complexes; (b)contacting each of the surface-bound complexes with two detectionreagents for the analyte to form a plurality of detection complexes,wherein the two detection reagents comprise a first proximity probe anda second proximity probe, respectively; (c) contacting each detectioncomplex formed in (b) with one or more connector oligonucleotidesincluding a first connector probe complementary to a first region of thefirst proximity probe and a first region on the second proximity probe,and a second connector probe complementary to a second non-overlappingregion of the first proximity probe and a second non-overlapping regionof the second proximity probe, the contacting step (c) is performedunder conditions sufficient to ligate the first and second proximityprobes to form a plurality of target sequences; (d) amplifying theplurality of target sequences to generate a plurality of amplicons eachcomprising a detection sequence; (e) hybridizing a plurality ofdetection probes to the plurality of detection probe sequences; and (f)measuring the amount of analytes bound to the surface.

Another embodiment is a method of detecting a plurality of analytes ofinterest in a sample comprising: (a) contacting the sample with asurface comprising a plurality of discrete binding domains, each bindingdomain comprising a binding reagent for an analyte, wherein thecontacting step forms a plurality of surface-bound complexes; (b)contacting each of the surface-bound complexes with two detectionreagents for the analyte to form a plurality of detection complexes,wherein the two detection reagents comprise a first proximity probe anda second proximity probe, respectively; (c) contacting each detectioncomplex formed in (b) with one or more connector oligonucleotidesincluding a first circularization probe complementary to a first regionof the first proximity probe and a first region on the second proximityprobe, and a second circularization probe complementary to a secondnon-overlapping region of the first proximity probe and a secondnon-overlapping region of the second proximity probe, the contactingstep (c) is performed under conditions sufficient to form a plurality ofcircular DNA templates; (d) amplifying the plurality of circular DNAtemplates to generate a plurality of amplicons each comprising aplurality of detection sequences; (e) hybridizing a plurality ofdetection probes to the plurality of detection probe sequences; and (f)measuring the amount of analytes bound to the surface.

In addition, the invention includes a kit for the measurement of ananalyte of interest in a sample, the kit comprising: (a) a surfacecomprising a binding reagent for the analyte; and (b) in one or morecontainers, compartments, or vessels: (i) two detection reagents for theanalyte, wherein the two detection reagents comprise a first proximityprobe and a second proximity probe, respectively; (ii) one or moreconnector oligonucleotides including a first connector probecomplementary to a first region of the first proximity probe and a firstregion on the second proximity probe, and a second connector probecomplementary to a second non-overlapping region of the first proximityprobe and a second non-overlapping region of the second proximity probe;and (iii) one or more detection probes complementary to the detectionprobe sequence.

Also provided is a kit for the measurement of an analyte of interest ina sample, the kit comprising: (a) a surface comprising a binding reagentfor the analyte; and (b) in one or more containers, compartments, orvessels: (i) two detection reagents for the analyte, wherein the twodetection reagents comprise a first proximity probe and a secondproximity probe, respectively; (ii) one or more connectoroligonucleotides including a first circularization probe complementaryto a first region of the first proximity probe and a first region on thesecond proximity probe, and a second circularization probe complementaryto a second non-overlapping region of the first proximity probe and asecond non-overlapping region of the second proximity probe; and (iii)one or more detection probes complementary to the detection probesequence.

Further provided is a kit for the measurement of a plurality of analytesof interest in a sample, the kit comprising: (a) a surface comprising aplurality of discrete binding domains, each binding domain comprising abinding reagent for an analyte; and (b) in one or more containers,compartments, or vessels: (i) two detection reagents for each analyte,wherein the two detection reagents comprise a first proximity probe anda second proximity probe, respectively; (ii) one or more connectoroligonucleotides including a first circularization probe complementaryto a first region of the first proximity probe and a first region on thesecond proximity probe, and a second circularization probe complementaryto a second non-overlapping region of the first proximity probe and asecond non-overlapping region of the second proximity probe; and (iii)one or more detection probes complementary to the detection probesequence.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1( a)-(b) illustrate the formation of a sandwich immunoassaycomplex and the attachment of an amplified oligonucleotide product thatcontains multiple detection labeling sites, thereby amplifying thedetectable signal for each individual binding event. FIG. 1( b) showsthe addition of an anchoring reagent including an anchoringoligonucleotide sequence that is complementary to a sequence of theamplicon that forms as the assay method progresses.

FIG. 2 shows one method of attaching an oligonucleotide to a protein.

FIG. 3( a) illustrates a preferred embodiment of the invention in whicha surface bound complex is formed between a capture reagent, theanalyte, and two detection reagents, each attached to a first and secondproximity probe, respectively, which are ligated to connector probes toform a circular DNA template that is amplified by rolling circleamplification. Circ-1, SEQ ID NO: 4; Circ-2, SEQ ID NO: 5; PP1, SEQ IDNO: 1 (poly-A tail truncated); PP2, SEQ ID NO: 2. FIG. 3( a) alsoincludes an amplification reagent that includes an anchoringoligonucleotide sequence that is complementary to a sequence of theamplicon that forms as the assay method progresses. FIG. 3( b) shows anexemplary sequence (SEQ ID NO: 3) of the first circular DNA templateCirc-1, a detection oligonucleotide sequence, the inert region of theamplicon, and a portion PP2, which is designed to hybridize to thesecond proximity probe. FIG. 3( c) depicts an embodiment with Circ-1(SEQ ID NO: 3) and Circ-2 (SEQ ID NO: 4) hybridized to PP1 (SEQ IDNO: 1) and PP2 (SEQ ID NO: 2). In this alternative embodiment, theanchoring reagent is omitted.

FIGS. 4 and 5( a)-(b) illustrate alternative methods of generating anamplicon that can be amplified by rolling circle amplification.

FIG. 6 illustrates an alternative embodiment in which a portion of eachof the proximity probes in the sandwich complex is temporarily protectedby short strands of RNA hybridized to each segment. Those strands areenzymatically removed to allow the proximity probes to hybridize to oneanother and the chain to be extended.

FIG. 7 shows a further embodiment in which proximity probes are attachedto the capture reagent and a detection reagent, and a portion of eachproximity probe is temporarily protected by short strands of RNAhybridized thereto, as described above in reference to FIG. 6.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The articles“a” and “an” are used herein to refer to one or to more than one (i.e.,to at least one) of the grammatical object of the article. By way ofexample, “an element” means one element or more than one element.

The present invention is designed to improve immunoassay methods byamplifying the signal from labeled antigen-antibody complexes andoptionally, anchoring that complex to overcome instability that mayarise from low antibody affinity and/or the attachment of a highmolecular weight label or labeling site. The invention includes methodsthat improve on existing techniques by (i) attaching an amplifiedoligonucleotide product that contains multiple detection labeling sitesto the sandwich immunoassay complex formed in a sandwich immunoassay,thereby amplifying the detectable signal for each individual bindingevent, and optionally, (ii) anchoring the sandwich immunoassay complexand the attached amplicon. In a preferred embodiment, the methodincludes attaching an amplified oligonucleotide product that includesmultiple detection labeling sites to the sandwich immunoassay complex,and anchoring the complex to the surface to ensure that the complex isretained on the surface. This modified immunoassay method can be used todetect extremely low numbers of binding events, even individualantigen-antibody complexes. The basic approach is not limited toimmunoassays and can be used to carry out binding assays using otherclasses of binding reagents

One embodiment of the invention is illustrated in FIG. 1( a). Thebinding surface (101) includes a capture reagent (102) that bindsanalyte, A, and an adjacent anchoring reagent (103) that includes ananchoring oligonucleotide sequence (104). The anchoring sequence isdesigned to be complementary to a portion of an amplicon that isextended from the sandwich immunoassay complex as the assay methodprogresses. The binding surface is contacted with a sample containingthe analyte, which then binds to the surface-bound capture reagent. Thesurface is also contacted with one or more detection reagents (105 and106) that bind analyte A.

In a preferred embodiment, two detection reagents, a first detectionreagent (105) and a second detection reagent (106) are added, which bindto the surface bound analyte to form a sandwich complex (109) in whichthree binding reagents: the capture reagent and two detection reagentsare bound to the analyte. The first detection reagent includes a firstoligonucleotide probe (referred to as the first proximity probe (107))and the second detection reagent includes a second oligonucleotide probe(referred to as the second proximity probe (108)). The proximity probesare designed such that the first proximity probe is extended underspecified amplification conditions, but only when both proximity probesare present in the sandwich complex.

In one embodiment, a proximity ligation amplification (PLA) is carriedout to extend the first proximity probe. The sandwich complex comprisingthe two proximity probes is contacted with one or more connectoroligonucleotides/probes (110). Preferably, the connector probes includea first connector probe that hybridizes to a first region on the firstproximity probe and a first region on the second proximity probe; and asecond connector probe that hybridizes to a second non-overlappingregion of the first proximity probe and a second non-overlapping regionof the second proximity probe. Ligation of hybridized connectorsequences forms a circular oligonucleotide that is then used to extendthe first proximity probe by rolling circle amplification (RCA) of thecircle. Suitable probe designs and amplification conditions forproximity ligation amplification are well established in the art. Aunique aspect of the present invention is the inclusion in one of theconnector probes of the same sequence as is used in the anchoringreagent. During extension of the first proximity probe, the extendedregion thereby includes the complement of the anchoring sequence, whichhybridizes to the anchoring reagent, thereby stabilizing the sandwichcomplex and preventing dissociation of the first proximity probe. Theextended first proximity probe may contain detectable labels (e.g., byinclusion of labeled nucleotides during the RCA extension reaction) thatcan be measured to measure the amount of analyte on the surface.Alternatively, a plurality of labeled probes comprising detectablelabels are added and hybridized to the extended first proximity probe,and the amount of analyte bound to the surface is measured. Analternative embodiment of the invention is illustrated in FIG. 1( b). Inthis embodiment, the anchoring reagent is omitted.

The skilled artisan in the field of binding assays will readilyappreciate the scope of binding reagents and companion binding partnersthat may be used in the present methods. A non-limiting list of suchpairs include (in either order) receptor/ligand pairs,antibodies/antigens, natural or synthetic receptor/ligand pairs, aminesand carbonyl compounds (i.e., binding through the formation of aSchiff's base), hapten/antibody pairs, antigen/antibody pairs,epitope/antibody pairs, mimitope/antibody pairs, aptamer/target moleculepairs, hybridization partners, and intercalater/target molecule pairs.In one embodiment, the binding assays employ antibodies or otherreceptor proteins as binding reagents. The term “antibody” includesintact antibody molecules (including hybrid antibodies assembled by invitro re-association of antibody subunits), antibody fragments andrecombinant protein constructs comprising an antigen binding domain ofan antibody (as described, e.g., in Porter, R. R. and Weir, R. C. J.Cell Physiol., 67 (Suppl); 51-64 (1966) and Hochman, 1. Inbar, D. andGivol, D. Biochemistry 12: 1130 (1973)), as well as antibody constructsthat have been chemically modified, e.g., by the introduction of adetectable label.

The anchoring reagent includes an anchoring sequence that is directly orindirectly bound (e.g., through binding reactions) to the surface, e.g.,using methods established in the art for immobilizing oligonucleotides.In one embodiment, the anchoring reagent comprises a protein linked orotherwise bound to the anchoring sequence. In this embodiment, anyprotein can be used that can be immobilized on a surface and modified byan anchoring oligonucleotide. Non-limiting examples includestreptavidin, avidin, or bovine serum albumin (BSA). In a preferredembodiment, the anchoring reagent comprises BSA. The protein can bemodified by an anchoring oligonucleotide and attached to a surface usingknown methods, e.g., as illustrated in FIG. 2, usingsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC), a well-established heterobifunctional cross-linking agent.Reaction of the N-hydroxysuccinimide (NHS) group of SMCC with bovineserum albumin (BSA) labels the BSA with thiol-reactive maleimide groups.The maleimide groups are, in turn, reacted with thiol-modifiedoligonucleotides to form BSA-oligonucleotide conjugates that are linkedthrough stable thioether bonds. In one specific example, arrays areformed by printing arrays of the BSA-oligonucleotide conjugates ongraphitic carbon surfaces, preferably screen printed carbon inkelectrodes. Alternatively, if the protein is avidin or streptavidin, theanchoring sequence can be linked to biotin and joined to immobilizedavidin or streptavidin through biotin-avidin or biotin-streptavidininteractions.

The anchoring oligonucleotide attached to the anchoring reagent can beany sequence that will hybridize to the amplicon that develops duringthe amplification process. In a preferred embodiment, the anchoringoligonucleotide sequence comprises a poly(A) sequence attached to theanchor sequence complementary to an inert region of the ampliconsequence or a portion thereof. In one embodiment, a sequence that iscomplementary to the full length of the inert region of the amplicon isincluded, about 25 nucleotides in length), alone or in combination witha poly(A) arm of e.g., up to 30 nucleotides in length. Preferably, theanchoring oligonucleotide is selected from: (i) (full length complementto the inert region of the amplicon, 25 nucleotides in length)-(20nucleotide poly (A) arm); or (ii) (complement to a portion of the inertregion of the amplicon, 15 nucleotides in length)-(30 nucleotide poly(A) arm).

Any suitable amplification technique can be used to generate theamplicon, including but not limited to, PCR (Polymerase Chain Reaction),LCR (Ligase Chain Reaction), SDA (Strand Displacement Amplification),3SR (Self-Sustained Synthetic Reaction), and isothermal amplificationmethods, e.g., helicase-dependent amplification and rolling circleamplification (RCA). In a preferred embodiment, RCA is used because ithas significant advantages in terms of sensitivity, multiplexing,dynamic range and scalability. Techniques for RCA are known in the art(see, e.g., Baner et al, Nucleic Acids Research, 26:5073 5078, 1998;Lizardi et al., Nature Genetics 19:226, 1998; Schweitzer et al. Proc.Natl. Acad. Sci. USA 97:10113 119, 2000; Faruqi et al., BMC Genomics2:4, 2000; Nallur et al., Nucl. Acids Res. 29:e118, 2001; Dean et al.Genome Res. 11:1095 1099, 2001; Schweitzer et al., Nature Biotech.20:359 365, 2002; U.S. Pat. Nos. 6,054,274, 6,291,187, 6,323,009,6,344,329 and 6,368,801). Several different variants of RCA are known,including linear RCA (LRCA) and exponential RCA (ERCA). RCA generatesmany thousands of copies of a circular template, with the chain ofcopies attached to the original target DNA, allowing for spatialresolution of target and rapid amplification of the signal. RCAfacilitates (i) detection of single target molecules; (ii) amplificationof signals from proteins as well as DNA and RNA; (iii) identifying thelocation of molecules that have been amplified on a solid surface; (iv)measurement of many different targets simultaneously; and (v) analysisof one or more targets in solution or solid phase.

A specific embodiment of the invention is depicted in FIG. 3( a). Acomplex is formed on a surface (301) between a capture reagent (302),the analyte (303) and two detection reagents (304 and 305), eachincluding a first proximity probe (306) (PP1, SEQ ID NO: 1) and a secondproximity probe (307) (PP2, SEQ ID NO: 2), respectively. First andsecond connector oligonucleotides Circ-1 (308) (SEQ ID NO: 3) and Circ-2(309) (SEQ ID NO: 4), respectively, in FIG. 3( a)) are added, which whenboth proximity probes are present in the complex, each hybridize to andbridge the two proximity probes. The bound connector probes are ligatedat ligations sites 1 and 2 (310 and 311), respectively to form acircular DNA template (312). The circular DNA template is amplified byrolling circle amplification to extend the first proximity probe and,thereby, generate an amplicon comprising a plurality of detectionsequences (313) and an anchoring oligonucleotide sequence complement(314) (including a partial anchoring sequence complement (315)). Theanchoring oligonucleotide sequence (316) (attached to a capture moiety(317)) and its complement hybridize, a plurality of detection probes arehybridized to the plurality of detection probe sequences, and the amountof analyte bound to the surface is measured (not shown but illustratedin FIG. 1( a)). FIG. 3( b) shows an exemplary sequence of the firstcircular DNA template Circ-1 (308) (SEQ ID NO: 3), which is designed tohybridize to the first proximity probe (PP1), a detectionoligonucleotide sequence, the inert region of the amplicon (which can beused in whole or in part to bind to the anchoring oligonucleotidesequence), and a portion PP2 (which is designed to hybridize to thesecond proximity probe). An alternative embodiment is depicted in FIG.3( c). A complex is formed on a surface (318) between a binding reagent(319), the analyte (320) and two detection reagents (321 and 322), eachincluding a first and second proximity probe (323 and 324),respectively. First and second circularization oligonucleotides (Circ-1(325) (SEQ ID NO: 3) and Circ-2 (326) (SEQ ID NO: 4), respectively inFIG. 3( c)) are added which ligate to the proximity probe sequences atligations sites 1 and 2 (327 and 328), respectively and extended to forma circular DNA template (329). The circular DNA template is amplified byrolling circle amplification to generate an amplicon comprising aplurality of detection sequences (330). A plurality of detection probesare hybridized to the plurality of detection probe sequences, and theamount of analyte bound to the surface is measured. It is to beunderstood that the poly-A tails of proximity probes PP1 and PP2 asdisclosed in the Figures, such as FIG. 3( c), may vary in the number ofAlanine repeats specifically shown in the Figures, relative to thedisclosed PP 1 and/or PP2 sequences, without intending to alter thesequences disclosed herein.

Another approach to generating a target sequence that is amplified byRCA or any suitable amplification method is illustrated in FIG. 4. Inthis embodiment, each of the proximity probes can fold into a loopedhairpin structure. The formation of these hairpin structures generates asingle stranded loop and double stranded portion containing arecombination signal. Recombinase is added drive the recombination ofthe two hairpin structures to form a circular DNA template, which issubsequently subjected to RCA as described above. The amplicon islabeled and optionally anchored to an anchoring reagent and analyte isdetected. The key element of this embodiment is the ability ofrecombinases to catalyze the site specific recombination of DNAcontaining sequence specific recombination sites. For example, CreRecombinase from the bacteriophage P1 catalyzes recombination at sitescontaining loxP sites and other non-limiting examples include but arenot limited to Flippase (flp, from Yeast), Hin (Salmonella), and Tre, anengineered (evolved) version of Cre. This alternative approach does notrequire the addition of additional components such as oligonucleotidetemplates, ATP and dNTPs. In this embodiment, the loxP (recombination)sites are preferably modified to be non-symmetrical, resulting in ashift in the normal equilibrium towards the formation of the desiredrecombined product. This is illustrated in FIG. 4, with the light/darkshading of the recombination sites.

Moreover, FIG. 5( a) illustrates yet another method to generate a targetsequence that is amplified by RCA or any suitable amplification method.Each of the proximity probes attached to the detection reagents includea loxP site that enables site specific recombination between the twooligonucleotides by Cre recombinase, resulting in the formation of a newoligonucleotide sequence that is composed of the 5′ portion of oneproximity probe and the 3′ portion of the other proximity probe, thatflank the lox P sites. The newly created target sequence can besubsequently amplified by any suitable method, labeled, optionallyanchored, and detected as described above. FIG. 5( a) illustrates thisembodiment using the T7 RNA polymerase promoter as the operable elementfor amplification. It will also be understood that other RNA polymerasesites such as T3 and SP6 linked at either the 3 or 5′ portions of theproximity probes, are equally suitable for use in this method. In thisembodiment, the loxP (recombination) sites are preferably modified to benon-symmetrical, resulting in a shift in the normal equilibrium towardsthe formation of the desired recombined product. As shown in FIG. 5( b),the method can also be used to generate a circular DNA template that canbe used in RCA.

The various reagents employed in the method can be combined in one ormore additional or alternative steps without departing from the spiritor scope of the invention. For example, the reagents can be combinedsequentially, e.g., analyte is added to the binding reaction on thesurface, followed by the addition of detection reagent(s), and thenamplification, and detection, such that the method is carried out on thesurface. Alternatively, one or more reagents can be combined in solutionand then contacted with the surface. For example, the analyte can becombined with the detection reagents and then contacted with thesurface, followed by amplification and detection, and/or the detectionreagents can be combined with the analyte, the PLA amplification carriedout in solution, and the resulting complex contacted by the surface tocarry out the remaining steps of the methods.

Examples of samples that may be analyzed by the methods of the presentinvention include, but are not limited to food samples (including foodextracts, food homogenates, beverages, etc.), environmental samples(e.g., soil samples, environmental sludges, collected environmentalaerosols, environmental wipes, water filtrates, etc.), industrialsamples (e.g., starting materials, products or intermediates from anindustrial production process), human clinical samples, veterinarysamples and other samples of biological origin. Biological samples thatmay be analyzed include, but are not limited to, feces, mucosal swabs,physiological samples and/or samples containing suspensions of cells.Specific examples of biological samples include blood, serum, plasma,feces, mucosal swabs, tissue aspirates, tissue homogenates, cellcultures and cell culture supernatants (including cultures of eukaryoticand prokaryotic cells), urine, saliva, sputum, and cerebrospinal sample.

Analytes that may be measured using the methods of the inventioninclude, but are not limited to proteins, toxins, nucleic acids,microorganisms, viruses, cells, fungi, spores, carbohydrates, lipids,glycoproteins, lipoproteins, polysaccharides, drugs, hormones, steroids,nutrients, metabolites and any modified derivative of the abovemolecules, or any complex comprising one or more of the above moleculesor combinations thereof. The level of an analyte of interest in a samplemay be indicative of a disease or disease condition or it may simplyindicate whether the patient was exposed to that analyte.

The assays of the present invention may be used to determine theconcentration of one or more, e.g., two or more analytes in a sample.Thus, two or more analytes may be measured in the same sample. Panels ofanalytes that can be measured in the same sample include, for example,panels of assays for analytes or activities associated with a diseasestate or physiological conditions. Certain such panels include panels ofcytokines and/or their receptors (e.g., one or more of TNF-alpha,TNF-beta, IL1-alpha, IL1-beta, IL2, IL4, IL6, IL-10, IL-12, IFN-y,etc.), growth factors and/or their receptors (e.g., one or more of EGF,VGF, TGF, VEGF, etc.), drugs of abuse, therapeutic drugs, vitamins,pathogen specific antibodies, auto-antibodies (e.g., one or moreantibodies directed against the Sm, RNP, SS-A, SS-alpha, J0-1, andSc1-70 antigens), allergen-specific antibodies, tumor markers (e.g., oneor more of CEA, PSA, CA-125 II, CA 15-3, CA 19-9, CA 72-4, CYFRA 21-1,NSE, AFP, etc.), markers of cardiac disease including congestive heartdisease and/or acute myocardial infarction (e.g., one or more ofTroponin T, Troponin I, myoglobin, CKMB, myeloperoxidase, glutathioneperoxidase, β-natriuretic protein (BNP), alpha-natriuretic protein(ANP), endothelin, aldosterone, C-reactive protein (CRP), etc.), markersassociated with hemostasis (e.g., one or more of Fibrin monomer,D-dimer, thrombin-antithrombin complex, prothrombin fragments 1 & 2,anti-Factor Xa, etc.), markers of acute viral hepatitis infection (e.g.,one or more of IgM antibody to hepatitis A virus, IgM antibody tohepatitis B core antigen, hepatitis B surface antigen, antibody tohepatitis C virus, etc.), markers of Alzheimers Disease (alpha-amyloid,beta-amyloid, Aβ42, Aβ40, Aβ38, Aβ39, Aβ37, Aβ34, tau-protein, etc.),markers of osteoporosis (e.g., one or more of cross-linked NorC-telopeptides, total deoxypyridinoline, free deoxypyridinoline,osteocalcin, alkaline phosphatase, C-terminal propeptide of type Icollagen, bone-specific alkaline phosphatase, etc.), markers offertility state or fertility associated disorders (e.g., one or more ofEstradiol, progesterone, follicle stimulating hormone (FSH), lutenizinghormone (LH), prolactin, hCG, testosterone, etc.), markers of thyroiddisorders (e.g., one or more of thyroid stimulating hormone (TSH), TotalT3, Free T3, Total T4, Free T4, and reverse T3), and markers of prostatecancer (e.g., one or more of total PSA, free PSA, complexed PSA,prostatic acid phosphatase, creatine kinase, etc.). Certain embodimentsof invention include measuring, e.g., one or more, two or more, four ormore or 10 or more analytes associated with a specific disease state orphysiological condition (e.g., analytes grouped together in a panel,such as those listed above; e.g., a panel useful for the diagnosis ofthyroid disorders may include e.g., one or more of thyroid stimulatinghormone (TSH), Total T3, Free T3, Total T4, Free T4, and reverse T3).

The methods of the present invention are designed to allow detection ofa wide variety of biological and biochemical agents, as described above.In one embodiment, the methods may be used to detect pathogenic and/orpotentially pathogenic virus, bacteria and toxins including biologicalwarfare agents (“BWAs”) in a variety of relevant clinical andenvironmental matrices, including and without limitation, blood, sputum,stool, filters, swabs, etc. A non-limiting list of pathogens and toxinsthat may be analyzed (alone or in combination) using the methods of thepresent invention is Bacillus anthracia (anthrax), Yersinia pestis(plague), Vibrio cholerae (cholera), Francisella tularensis (tularemia),Brucella spp. (Brucellosis), Coxiella burnetii (Q fever), orthopoxviruses including variola virus (smallpox), viral encephalitis,Venezuelan equine encephalitis virus (VEE), western equine encephalitisvirus (WEE), eastern equine encephalitis virus (EEE), Alphavirus, viralhemorrhagic fevers, Arenaviridae, Bunyaviridae, Filoviridae,Flaviviridae, Ebola virus, staphylococcal enterotoxins, ricin, botulinumtoxins, Clostridium botulinum, mycotoxin, Fusarium, Myrotecium,Cephalosporium, Trichoderma, Verticimonosporium, Stachybotrys, glanders,wheat fungus, Bacillus globigii, Serratia marcescens, yellow rain,trichothecene mycotoxins, Salmonella typhimurium, aflatoxin, Xenopsyllacheopis, Diamanus montanus, alastrim, monkeypox, Arenavirus, Hantavirus,Lassa fever, Argentine hemorrhagic fevers, Bolivian hemorrhagic fevers,Rift Valley fever virus, Crimean-Congo virus, Hanta virus, Marburghemorrhagic fevers, yellow fever virus, dengue fever viruses, influenza(including human and animal strains including H5N1 avian influenza),human immunodeficiency viruses I and II (HIV I and II), hepatitis A,hepatitis B, hepatitis C, hepatitis (non-A, B or C), Enterovirus,Epstein-Barr virus, Cytomegalovirus, herpes simplex viruses, Chlamydiatrachomatis, Neisseria gonorrheae, Trichomonas vaginalis, humanpapilloma virus, Treponema pallidum, Streptococcus pneumonia,Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae,Legionella pneumophila, Staphylococcus aureus, Moraxella catarrhalis,Streptococcus pyogenes, Clostridium difficile, Neisseria meningitidis,Klebsiella pneumoniae, Mycobacterium tuberculosis, coronavirus,Coxsackie A virus, rhinovirus, parainfluenza virus, respiratorysyncytial virus (RSV), metapneumovirus, and adenovirus.

A wide variety of solid phases are suitable for use in the methods ofthe present invention including conventional solid phases from the artof binding assays. Solid phases may be made from a variety of differentmaterials including polymers (e.g., polystyrene and polypropylene),ceramics, glass, composite materials (e.g., carbon-polymer compositessuch as carbon-based inks). Suitable solid phases include the surfacesof macroscopic objects such as an interior surface of an assay container(e.g., test tubes, cuvettes, flow cells, cartridges, wells in amulti-well plate, etc.), slides, assay chips (such as those used in geneor protein chip measurements), pins or probes, beads, filtration media,lateral flow media (for example, filtration membranes used in lateralflow test strips), etc.

Suitable solid phases also include particles (including but not limitedto colloids or beads) commonly used in other types of particle-basedassays e.g., magnetic, polypropylene, and latex particles, materialstypically used in solid-phase synthesis e.g., polystyrene andpolyacrylamide particles, and materials typically used inchromatographic applications e.g., silica, alumina, polyacrylamide,polystyrene. The materials may also be a fiber such as a carbon fibril.Microparticles may be inanimate or alternatively, may include animatebiological entities such as cells, viruses, bacterium and the like. Aparticle used in the present method may be comprised of any materialsuitable for attachment to one or more binding reagents, and that may becollected via, e.g., centrifugation, gravity, filtration or magneticcollection. A wide variety of different types of particles that may beattached to binding reagents are sold commercially for use in bindingassays. These include non-magnetic particles as well as particlescomprising magnetizable materials which allow the particles to becollected with a magnetic field. In one embodiment, the particles arecomprised of a conductive and/or semiconductive material, e.g.,colloidal gold particles. The microparticles may have a wide variety ofsizes and shapes. By way of example and not limitation, microparticlesmay be between 5 nanometers and 100 micrometers. Preferablymicroparticles have sizes between 20 nm and 10 micrometers. Theparticles may be spherical, oblong, rod-like, etc., or they may beirregular in shape.

The methods of the present invention may be used in a variety of assaydevices and/or formats. The assay devices may include, e.g., assaymodules, such as assay plates, cartridges, multi-well assay plates,reaction vessels, test tubes, cuvettes, flow cells, assay chips, lateralflow devices, etc., having assay reagents (which may include targetingagents or other binding reagents) added as the assay progresses orpre-loaded in the wells, chambers, or assay regions of the assay module.These devices may employ a variety of assay formats for specific bindingassays, e.g., immunoassay or immunochromatographic assays. Illustrativeassay devices and formats are described herein below. In certainembodiments, the methods of the present invention may employ assayreagents that are stored in a dry state and the assay devices/kits mayfurther comprise or be supplied with desiccant materials for maintainingthe assay reagents in a dry state. The assay devices preloaded with theassay reagents can greatly improve the speed and reduce the complexityof assay measurements while maintaining excellent stability duringstorage. The dried assay reagents may be any assay reagent that can bedried and then reconstituted prior to use in an assay. These include,but are not limited to, binding reagents useful in binding assays,enzymes, enzyme substrates, indicator dyes and other reactive compoundsthat may be used to detect an analyte of interest. The assay reagentsmay also include substances that are not directly involved in themechanism of detection but play an auxiliary role in an assay including,but not limited to, blocking agents, stabilizing agents, detergents,salts, pH buffers, preservatives, etc. Reagents may be present in freeform or supported on solid phases including the surfaces of compartments(e.g., chambers, channels, flow cells, wells, etc.) in the assay modulesor the surfaces of colloids, beads, or other particulate supports.

The methods of the invention can be used with a variety of methods formeasuring the amount of an analyte and, in particular, measuring theamount of an analyte bound to a solid phase. Techniques that may be usedinclude, but are not limited to, techniques known in the art such ascell culture-based assays, binding assays (including agglutinationtests, immunoassays, nucleic acid hybridization assays, etc.), enzymaticassays, colorometric assays, etc. Other suitable techniques will bereadily apparent to one of average skill in the art. Some measurementtechniques allow for measurements to be made by visual inspection,others may require or benefit from the use of an instrument to conductthe measurement.

Methods for measuring the amount of an analyte include label-freetechniques, which include but are not limited to i) techniques thatmeasure changes in mass or refractive index at a surface after bindingof an analyte to a surface (e.g., surface acoustic wave techniques,surface plasmon resonance sensors, ellipsometric techniques, etc.), ii)mass spectrometric techniques (including techniques like MALDI, SELDI,etc. that can measure analytes on a surface), iii) chromatographic orelectrophoretic techniques, iv) fluorescence techniques (which may bebased on the inherent fluorescence of an analyte), etc.

Methods for measuring the amount of an analyte also include techniquesthat measure analytes through the detection of labels which may beattached directly or indirectly (e.g., through the use of labeledbinding partners of an analyte) to an analyte. Suitable labels includelabels that can be directly visualized (e.g., particles that may be seenvisually and labels that generate an measurable signal such as lightscattering, optical absorbance, fluorescence, chemiluminescence,electrochemiluminescence, radioactivity, magnetic fields, etc). Labelsthat may be used also include enzymes or other chemically reactivespecies that have a chemical activity that leads to a measurable signalsuch as light scattering, absorbance, fluorescence, etc. The use ofenzymes as labels has been well established in Enzyme-LinkedImmunoSorbent Assays, also called ELISAs, Enzyme ImmunoAssays or EIAs.In the ELISA format, an unknown amount of antigen is affixed to asurface and then a specific antibody is washed over the surface so thatit can bind to the antigen. This antibody is linked to an enzyme, and inthe final step a substance is added that the enzyme converts to aproduct that provides a change in a detectable signal. The formation ofproduct may be detectable, e.g., due a difference, relative to thesubstrate, in a measurable property such as absorbance, fluorescence,chemiluminescence, light scattering, etc. Certain (but not all)measurement methods that may be used with solid phase binding methodsaccording to the invention may benefit from or require a wash step toremove unbound components (e.g., labels) from the solid phaseAccordingly, the methods of the invention may comprise such a wash step.

In one embodiment, an analyte(s) of interest in the sample may bemeasured using electrochemiluminescence-based assay formats, e.g.electrochemiluminescence (ECL) based immunoassays. The high sensitivity,broad dynamic range and selectivity of ECL are important factors formedical diagnostics. Commercially available ECL instruments havedemonstrated exceptional performance and they have become widely usedfor reasons including their excellent sensitivity, dynamic range,precision, and tolerance of complex sample matrices. Species that can beinduced to emit ECL (ECL-active species) have been used as ECL labels,e.g., i) organometallic compounds where the metal is from, for example,the noble metals of group VIII, including Ru-containing andOs-containing organometallic compounds such as thetris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminol and relatedcompounds. Species that participate with the ECL label in the ECLprocess are referred to herein as ECL coreactants. Commonly usedcoreactants include tertiary amines (e.g., see U.S. Pat. No. 5,846,485),oxalate, and persulfate for ECL from RuBpy and hydrogen peroxide for ECLfrom luminol (see, e.g., U.S. Pat. No. 5,240,863). The light generatedby ECL labels can be used as a reporter signal in diagnostic procedures(Bard et al., U.S. Pat. No. 5,238,808, herein incorporated byreference). For instance, an ECL label can be covalently coupled to abinding agent such as an antibody, nucleic acid probe, receptor orligand; the participation of the binding reagent in a bindinginteraction can be monitored by measuring ECL emitted from the ECLlabel. Alternatively, the ECL signal from an ECL-active compound may beindicative of the chemical environment (see, e.g., U.S. Pat. No.5,641,623 which describes ECL assays that monitor the formation ordestruction of ECL coreactants). For more background on ECL, ECL labels,ECL assays and instrumentation for conducting ECL assays see U.S. Pat.Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910; 5,641,623;5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434; 5,786,141;5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754; 5,240,863;6,207,369; 6,214,552 and 5,589,136 and Published PCT Nos. WO99/63347;WO00/03233; WO99/58962; WO99/32662; WO99/14599; WO98/12539; WO97/36931and WO98/57154, all of which are incorporated herein by reference.

The methods of the invention may be applied to singleplex or multiplexformats where multiple assay measurements are performed on a singlesample. Multiplex measurements that can be used with the inventioninclude, but are not limited to, multiplex measurements i) that involvethe use of multiple sensors; ii) that use discrete assay domains on asurface (e.g., an array) that are distinguishable based on location onthe surface; iii) that involve the use of reagents coated on particlesthat are distinguishable based on a particle property such as size,shape, color, etc.; iv) that produce assay signals that aredistinguishable based on optical properties (e.g., absorbance oremission spectrum) or v) that are based on temporal properties of assaysignal (e.g., time, frequency or phase of a signal).

According to one embodiment of the present invention, the methods of theinvention can be used to detect the presence of an analyte and/ordetermine the concentration of analyte molecules in a sample. In somecases, there is a correlation between the percentage of binding surfaces(e.g., binding domains) containing one or more analyte molecules and theconcentration of the analyte molecules in the sample. Thus, thequantification method of certain embodiments of the invention allows forcalculation of the number of analyte molecules in a sample based on thepercentage of binding surfaces that contain an analyte molecule. In someembodiments, the measure of the concentration of analyte molecules in asample will be determined using a calibration curve. Methods todetermine the concentration of analyte molecules in a sample arediscussed below.

Certain embodiments of present invention are distinguished by theability to detect and/or quantify low numbers/concentrations of analytemolecules in a sample. It is currently believed that this ability may beachieved by spatially isolating individual or small numbers of analytemolecules, for example, as when they are partitioned across an array ofbinding surfaces, and then detecting their presence in the bindingsurfaces. The presence of an analyte molecule in a binding surface canbe counted in a binary fashion (e.g., zero when an analyte molecule isabsent; one when an analyte molecule is present), for example bydetermining the presence of a detectable label in a binding surface thatcontains at least one analyte molecule.

In some embodiments, the plurality of analyte molecules may bepartitioned such that at least some of the binding surfaces contain noanalyte molecules and at least some binding surfaces contain at leastone or, in certain cases, only one analyte molecule. For example, insome cases, the plurality of analyte molecules may be partitioned suchthat a statistically significant fraction of the binding surfacescontain no analyte molecules and a statistically significant fraction ofbinding surfaces contain at least one analyte molecule. In other cases,the plurality of analyte molecules may be partitioned such that astatistically significant fraction of the binding surfaces contain noanalyte molecules and a statistically significant fraction of bindingsurfaces contain only one analyte molecule. In either case, the numberof the plurality of binding surfaces and/or fraction of the plurality ofbinding surfaces that contain or do not contain an analyte molecule maybe determined. The number and/or fraction of the plurality of bindingsurfaces that contain an analyte molecule can be related to theconcentration of analyte molecules in the sample. In some embodiments, ameasure of the concentration of analyte molecules in the sample isdetermined based on the determination of the number and/or fraction ofthe plurality of binding surfaces that contain an analyte molecule. Incertain such embodiments, the measure of the concentration of theanalyte molecules in the sample is determined at least in partcomparison of a measured parameter to a calibration standard and/or by aPoisson and/or Gaussian distribution analysis of the number or fractionof the plurality of binding surfaces that contain an analyte molecule,as discussed more below. A “statistically significant fraction” of thebinding surfaces that contain a specified quantity of dissociatedspecies is defined as the minimum number of binding surfaces that can bereproducibly determined to contain an analyte molecule with a particularsystem of detection (i.e., substantially similar results are obtainedfor multiple essentially identical samples comprising the target analytemolecule) and that is above the background noise (e.g., non-specificbinding) that is determined when carrying out the assay with a samplethat does not contain any analyte molecules, divided by the total numberof binding surfaces. The statistically significant fraction may beexperimentally determined for a certain assay type and equipment set up(e.g., for each analyte molecule determined, each binding ligand, etc).In certain embodiments, the percentage of binding surfaces (e.g., thestatistically significant fraction) which comprises only one or at leastone analyte molecule is less than about 10%, less than about 5%, lessthan about 1%, less than about 0.5%, or less than about 0.1% of thetotal binding surfaces. In some cases, the percentage of bindingsurfaces which do not contain an analyte molecule is at least about 20%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 75%, at least about 80%, at least about 90%,or at least about 95%, at least about 99%, at least about 99.5%, atleast about 99.9%, or greater, of the total number of binding surfaces.

In some embodiments, a measure of the concentration of analyte moleculesin the sample may be determined at least in part by comparison of ameasured parameter to a calibration standard. For example, the fractionof binding surfaces that comprise an analyte molecule may be comparedagainst a calibration curve to determine a measure of the concentrationof the analyte molecule in the sample. The calibration curve may beproduced by completing the assay with a plurality of standardizedsamples of known concentration under the conditions used to analyze thetest samples. A reading may be taken for the signal related to thedetection/quantification of the analyte molecules for each standardizedsample, therefore allowing for the formation of a calibration curverelating the detection of the analyte molecules with a knownconcentration of the analyte molecule. The assay may then be completedon a sample comprising the analyte molecule in an unknown concentration,and the detection of the analyte molecules from this assay may beplotted on the calibration curve, therefore determining a measure of theconcentration of the analyte molecule in the sample.

In some embodiments, the concentration of analyte molecules in thesample that may be substantially accurately determined is less thanabout 100 fM, less than about 10 fM, less than about 3 fM, less thanabout 1 fM, less than about 0.3 fM, less than about 0.1 fM, less thanabout 0.03 fM, or less. In some embodiments, the concentration ofanalyte molecules in the sample that may be substantially accuratelydetermined is between about 5000 fM and about 0.1 fM, between about 3000fM and about 0.1 fM, between about 1000 fM and about 0.1 fM, betweenabout 1000 fM and about 1 fM, between about 100 fM and about 1 fM,between about 100 fM and about 0.1 fM. The concentration of analytemolecules in a sample may be considered to be substantially accuratelydetermined if the measured concentration of the analyte molecules in thesample is within about 20% of the actual concentration of the analytemolecules in the sample. In certain embodiments, the measuredconcentration of the analyte molecules in the sample may be within about10%, within about 3%, or within about 1% of the actual concentration ofthe analyte molecules in the sample. The accuracy of the assay methodmay be determined, in some embodiments, by determining the concentrationof analyte molecules in a sample of a known concentration using theselected assay method.

One advantageous aspect of the methods of the invention, especially whencoupled to a sensitive optical detection technique is that the signalamplification allows for the detection of individual binding event asbright points of light. Quantitation of signal, can then be carried outby counting the individual events (which can provide better sensitivityfor low analyte concentrations by providing improved discrimination ofbinding events from background noise) or by integrating over the signalfor all binding events (which can provide better dynamic range formeasuring high analyte concentrations).

In the specific case of using an imaging technique to measure an opticalsignal (such as fluorescence, chemiluminescence orelectrochemiluminescence) a binding event can be detected as a brightpoint source of light. When the surface density of point sources is low(e.g., when the probability of finding a point source in an R×Rarea—where R is the spatial resolution of the detection system—is lessthan 10%), it is likely that any observed point source is due to asingle binding event. Under these conditions, counting events is likelyto provide the most sensitive measurement. As the surface densityincreases, it becomes increasingly likely that it will not be possibleto resolve and count individual binding events. Under these conditions,integrating the optical signal over the binding surface is likely toprovide a more accurate measurement.

In one embodiment, the invention provides a kit comprising (a) surfaceincluding a binding reagent for the analyte and an anchoring reagentcomprising an anchoring oligonucleotide complementary to an ampliconsequence; and (b) in one or more containers, compartments, or vessels:(ii) two detection reagents for the analyte, wherein the two detectionreagents comprise a first proximity probe and a second proximity probe,respectively; (ii) one or more connector oligonucleotides including afirst connector probe complementary to a first region of the firstproximity probe and a first region on the second proximity probe, and asecond connector probe complementary to a second non-overlapping regionof the first proximity probe and a second non-overlapping region of thesecond proximity probe; and (iii) one or more detection probescomplementary to the detection probe sequence.

In an additional embodiment, the invention provides a kit comprising (a)surface including a binding reagent for the analyte and a anchoringmoiety comprising an anchoring oligonucleotide complementary to anamplicon sequence; and (b) in one or more containers, compartments, orvessels: (ii) two detection reagents for the analyte, wherein the twodetection reagents comprise a first proximity probe and a secondproximity probe, respectively; (ii) one or more connectoroligonucleotides including a first circularization probe complementaryto a first region of the first proximity probe and a first region on thesecond proximity probe, and a second circularization probe complementaryto a second non-overlapping region of the first proximity probe and asecond non-overlapping region of the second proximity probe, thecontacting step (c) is performed under conditions sufficient to form acircular DNA template; and (iii) one or more detection probescomplementary to the detection probe sequence.

The kit can further include one or more of the following: one or moreadditional reagents, buffers, polymerase, ligase, and/or dNTPs. Inaddition, if the one or more detection probes comprise a detectable ECLlabel, the kit can also include an ECL co-reactant.

Additional Alternative Embodiments

A further embodiment is illustrated in FIG. 6. A portion of each of theproximity probes in the sandwich immunoassay complex in panel (a) aretemporarily protected by short strands of RNA hybridized to eachsegment. The RNA strands are enzymatically removed so that each of theproximity probes can hybridize to one another and the chain is extendedby polymerase extension using biotinylated dNTPs (panel (b)). Eachbiotinylated base incorporated into the chain is bound to streptavidinlabeled with a detectable label (panel (c)).

Yet another approach is illustrated in FIG. 7. Proximity probes can beattached to the anchoring reagent and a detection reagent (as shown inpanel (a)) or each of the proximity probes can be attached to twodetection reagents as described hereinabove (not shown). Much like themethod illustrated in FIG. 6, a portion of each of the proximity probesare temporarily protected by short strands of RNA hybridized to eachsegment. The RNA strands are enzymatically removed so that each of theproximity probes can hybridize to one another and the chain is extendedby polymerase extension using biotinylated dNTPs (panel (b)). Eachbiotinylated base incorporated into the chain is bound to streptavidinlabeled with a detectable label (panel (c)).

Examples General Protocol for Proximity Ligation and Rolling CircleAmplification

A pair of detection antibodies to a target analyte was modified by theaddition of proximity probes 1 and 2 as follows: to 20 ug firstdetection antibody in 10 uL buffer, 1 uL 4 mM sulfo-SMCC was added,diluted in DMSO, and incubated at room temperature for 2 hours. Thefinal concentration of the detection antibody was 2 mg/mL or higher.Three (3) uL of 100 uM thiol-modified oligonucleotide (proximity probe 1and 2) was reduced with 4 uL of 100 mM DTT in 50 uL of 55 mM phosphatebuffer, 150 mM NaCl, 20 mM EDTA, pH 7.0, for 1 hour at 37 C. Thesequences of proximity probes 1 and 2 are:

Thiol-modified proximity probe 1:SH-AAA AAA AAA AGA CGC TAA TAG TTA AGA CGC TTU UU(SEQ ID No. 1; wherein the three U resides are 2′ O-methyl RNA)Thiol-modified proximity probe 2: (SEQ ID No. 2)SH-AAA AAA AAA ATA TGA CAG AAC TAG ACA CTT TT.

Excess sulfo-SMCC and DTT were removed, e.g., by using three spin columnseparates and antibody and DNA were pooled for covalent conjugation. Thesolution was dialyzed against 1×PBS at 4 C overnight. Antibody-proximityprobe conjugates were purified, e.g, by gel filtration to removeunconjugated antibodies and oligonucleotides.

An MSD MULTI-SPOT® plate was blocked for 1 hour with appropriate MSD®blocking solution and washed. Each binding domain on the plate includeda anchoring antibody and anchoring moiety, BSA, to which anoligonucleotide specific for an amplicon is covalently attached.Twenty-five (25) μl assay diluent, calibrator, or sample (diluted asappropriate) was added to each well. The plate was incubated withshaking for 1-3 hours and each well was washed. A solution of detectionantibodies labeled with proximity probes 1 and 2, prepared as describedabove, was added to each well, and incubated with shaking for 1-2 hours(alternatively, each individual detection antibody can be sequentiallyadded, with each addition followed by a 1 hour incubation). A ligationmix was added to each well including the following components: (i)circularization oligonucleotide 1 (125 nM), circularizationoligonucleotide 2 (125 nM), ligation buffer, ATP (1 mM), BSA (250ug/mL), Tween 20 (0.05%), NaCl (250 mM), T4 DNA ligase (0.05 U/uL),wherein the each of the circularization oligonucleotides are:

Circularization oligonucleotide 1: (SEQ ID No. 3)Phosphate-CTA TTA GCG TCC AGT GAA TGC GAG TCC GTCTAA GAG AGT AGT AGA GCA GCC GTC AAG AGT GTC TA.Circularization oligonucleotide 2: (SEQ ID No. 4)Phosphate-GTT CTG TCA TAT TTA AGC GTC TTA A.

The plate was incubated with the ligation mix for 30 minutes at 37 C,washed to remove excess circularization oligonucleotides, and incubatedwith RCA mixture for 1.5 hour at 37 C, wherein the RCA mixture containsBSA (250 ug/mL), RCA buffer, dNTP (250 uM of each), Tween 20 (0.05%)Phi29 DNA polymerase (0.125 U/ml). The plate was washed and a mixture ofdetection probes were added and incubated for 30 minutes at 37 C,wherein the detection probe mixture includes: BSA (250 ug/ml), sonicatedsalmon sperm DNA (2.5 ug/ml), 2×SSC, Tween 20 (0.05%), detection probes(6.25 nM). The detection probe is STAG-(SA+Biotin)-CAG TGA ATG CGA GTCCGT CT (SEQ ID No. 5). The plate was washed with 150 μl MSD read bufferand read immediately on MSD SECTOR® 6000 Reader (plates and readersupplied by Meso Scale Discovery, Rockville, Md.).

This general procedure was used to detect the following analytes:troponinin I, Akt (total), phospho-Akt (473), phospho-Akt (308), FluNPA, IL-12p40, IL-12p70, Abeta1-42, bridging and isotyping Ig assaysusing TNFalpha model system, bridging and isotyping Ig assays using HepB surface antigen, and bridging and isotyping Ig assays using Lyme C6.In general, the increase in ECL signal relative to a standard sandwichimmunoassay ranges from 0.2-100 times, average increase in backgroundsignal was from 0-10×, and the average improvement in LOD (limits ofdetection) was 2-30×. For certain assays tested, e.g., Troponin-1, Akt(total), IL-12p40, IL-12p70, and Abeta1-42, the presence of anchoringmoiety improved signal and/or dilution linearity.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of themethod in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims. Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

REFERENCES

-   1. U.S. Pat. No. 7,306,904-   2. U.S. Pat. No. 7,320,860-   3. U.S. Pat. No. 7,351,528-   4. U.S. Pat. No. 7,192,703-   5. U.S. Pat. No. 6,878,515-   6. Zhou et al., Genome Biology (2004), 5: R28-   7. Dean et al., Genome Research (2001), 11: 1095-1099-   8. Soderberg et al., Methods (2008), 45: 227-232-   9. Fredriksson et al., Nature Biotech (2002), 20: 473-477-   10. Fredriksson et al., Nature Methods (2007), 4(4): 327-329-   11. Vincent et al., EMBO Reports (2005), 5(8): 795-800-   12. Gajadjar et al., Biotechniques (1010), 48(22): 145-152-   13. Schallmeiner et al, Nature Methods (2007) 4(2): 135-137-   14. Ericsson et al., Nucl. Acids Research (2008), 36(8): e45-   15. Darmanis et al., Biotechniques (2007), 43: 443-450-   16. Dahl et al., Proc. Natl. Acad. Sci. (2004), 101(13): 4548-4553-   17. Weibrecht et al., Expert Rev. Proteomics (2010), 7(3): 401-409-   18. Spits et al., Nature Protocols (2005), 1(4): 1965-1970-   19. Nordengrahn et al., Vet. Microbio (2008), 127: 227-236-   20. Vuoriluoto et al., Mol. Oncology (2011), 5: 105-111-   21. Zhang et al., Clinica Chimica Acta (2006), 363: 61-70-   22. Andras et al., Mol. Biotech. (2001), 19: 29-44-   23. Schweltzer et al., Proc. Natl. Acad. Sci. (2000), 97(18):    10113-10119-   24. Jeong, et al., Cell. Mol. Life Sci. (2009), 66: 3325-3336-   25. Gill et al., Nucleosides, Nucleotides, and Nucleic Acids (2008),    27: 224-245-   26. Gullberg, et al., Current Op. in Biotech. (2003), 14: 82-86-   27. Gustafsdottir, et al., Clinical Chemistry (2006), 52(6):    1152-1160-   28. U.S. Patent Publication No. 20100075862-   29. U.S. Pat. No. 8,222,047-   30. U.S. Pat. No. 8,236,574-   31. U.S. Pat. No. 8,338,776-   32. U.S. Patent Publication No. 20110212537-   33. U.S. Patent Publication No. 20120196774-   34. U.S. Patent Publication No. 20120289428

1.-22. (canceled)
 23. A method of detecting a plurality of analytes ofinterest in a sample comprising: a. Contacting the sample with a surfacecomprising a plurality of discrete binding domains, each binding domaincomprising (i) a binding reagents for an analyte, and (ii) an anchoringreagent comprising an anchoring sequence complementary to an ampliconsequence, wherein the contacting step forms a plurality of surface-boundcomplexes; b. Contacting each of the surface-bound complexes with twodetection reagents for the analyte to form a plurality of detectioncomplexes, wherein the two detection reagents comprise a first proximityprobe and a second proximity probe, respectively; c. Contacting eachdetection complex formed in (b) with one or more connectoroligonucleotides including a first connector probe complementary to afirst region of the first proximity probe and a first region on thesecond proximity probe, and a second connector probe complementary to asecond non-overlapping region of the first proximity probe and a secondnon-overlapping region of the second proximity probe, the contactingstep (c) is performed under conditions sufficient to ligate the firstand second proximity probes to form a plurality of target sequences; d.Amplifying the plurality of target sequences to generate a plurality ofamplicons each comprising a detection sequence and an anchoring sequencecomplement; e. Hybridizing each anchoring sequence to the anchoringsequence complement; f. Hybridizing a plurality of detection probes tothe plurality of detection probe sequences; and g. Measuring the amountof analytes bound to the surface.
 24. A method of detecting a pluralityof analytes of interest in a sample comprising: a. Contacting the samplewith a surface comprising a plurality of discrete binding domains, eachbinding domain comprising (i) a binding reagent for an analyte, and (ii)an anchoring reagent comprising an anchoring sequence complementary toan amplicon sequence, wherein the contacting step forms a plurality ofsurface-bound complexes; b. Contacting each of the surface-boundcomplexes with two detection reagents for the analyte to form aplurality of detection complexes, wherein the two detection reagentscomprise a first proximity probe and a second proximity probe,respectively; c. Contacting each detection complex formed in (b) withone or more connector oligonucleotides including a first circularizationprobe complementary to a first region of the first proximity probe and afirst region on the second proximity probe, and a second circularizationprobe complementary to a second non-overlapping region of the firstproximity probe and a second non-overlapping region of the secondproximity probe, the contacting step (c) is performed under conditionssufficient to form a plurality of circular DNA templates; d. Amplifyingthe plurality of circular DNA templates to generate a plurality ofamplicons each comprising a plurality of detection sequences and ananchoring sequence complement; e. Hybridizing each anchoring sequence tothe anchoring sequence complement; f. Hybridizing a plurality ofdetection probes to the plurality of detection probe sequences; and g.Measuring the amount of analytes bound to the surface.
 25. The method ofany one of claims 23-24 wherein the two detection reagents are eachantibodies and the analyte is an antigen recognized by the antibodies.26. The method of any one of claims 23-24 wherein the anchoring reagentis selected from biotin, avidin, streptavidin, or albumin.
 27. Themethod of any one of claims 23-24 wherein the measuring step comprisesmeasuring optical absorbance, fluorescence, phosphorescence,chemiluminescence, light scattering or magnetism.
 28. The method of anyone of claims 23-24 wherein each of the one or more detection primerscomprise a detectable label.
 29. The method of claim 28 wherein thedetectable label is an ECL label and the measuring step comprisesmeasuring an ECL signal and correlating the signal with an amount ofanalyte in the sample.
 30. The method of claim 29 wherein the support isan electrode and the measuring step further comprises applying a voltagewaveform to the electrode to generate ECL.
 31. The method of claim 23wherein the method further comprises exposing the sample to a pluralityof binding domains under conditions so that at least one analytemolecule is anchored in at least some of the binding domains, whereineach binding domain is positioned within a well of a multi-well plateand each binding domain defines a binding surface that has a bindingreagent immobilized thereon; determining the presence or absence of ananalyte molecule in each binding domain so as to identify the number ofbinding domains that contain an analyte molecule and/or the number ofbinding domains that do not contain an analyte molecule; and determiningthe concentration of analyte molecules in the sample to be tested fromthe number of binding domains that contain and/or do not contain ananalyte molecule.
 32. The method of claim 31 wherein at least about 99%of the binding domains contain either zero or one analyte molecule. 33.The method of claim 31 wherein at least about 95% of the binding domainscontain either zero or one analyte molecule.
 34. The method of claim 31wherein at least about 80% of the binding domains contain either zero orone analyte molecule.
 35. The method of claim 31 wherein at least about50% of the binding domains contain either zero or one analyte molecule.36. The method of claim 31 wherein at least about 10% of the bindingdomains contain either zero or one analyte molecule.
 37. The method ofclaim 31 wherein at least about 1% of the binding domains contain eitherzero or one analyte molecule.
 38. The method of any one of claims 31 to37 wherein the concentration of analyte molecules in the sample isdetermined at least in part using a calibration curve, a Poissondistribution analysis and/or a Gaussian distribution analysis of thenumber of binding domains that contain at least one or one analytemolecule.
 39. The method of claim 23 further comprising partitioning atleast a portion of the analyte molecules in the sample into a pluralityof binding domains, so that, for substantially all of the bindingdomains, each binding domain contains either no analyte molecules or asingle analyte molecule; determining the presence or absence of ananalyte molecule in a plurality of binding domains to provide a fractionof the interrogated binding domains that contain an analyte molecule;and determining the concentration of analyte molecules in the samplefrom the fraction of interrogated binding domains that contain ananalyte molecule.
 40. The method of claim 23 further comprisingpartitioning the sample into a plurality of second, smaller samples ofequal volume so that at least some of the second, smaller samplescontain either a single analyte molecule or no analyte molecules,determining the presence or absence of an analyte molecule in at least asubset of the second samples so as to identify the fraction of secondsamples in the subset that contain an analyte molecule; and determiningthe concentration of analyte molecules in the sample to be tested fromthe fraction of second samples of the subset that contain the analytemolecules.
 41. The method of claim 23 further comprising contacting anarray with the sample such that the ratio of the number of analytemolecules in the sample contacted with the array to the number ofbinding domains in the array is less than 1:1; and determining thenumber of binding domains which contain an analyte molecule, wherein thearray comprises a multi-well plate including a plurality of wells,wherein each well comprising a plurality of binding domains.
 42. Themethod of claim 41 wherein the ratio of the number of analyte moleculesin the sample contacted with the array to the number of binding domainsin the array is less than about 1:5.
 43. The method of claim 41 whereinthe ratio of the number of analyte molecules in the sample contactedwith the array to the number of binding domains in the array is lessthan about 1:10.
 44. The method of claim 41 wherein the ratio of thenumber of analyte molecules in the sample contacted with the array tothe number of binding domains in the array is less than about 1:100. 45.The method of claim 41 wherein the ratio of the number of analytemolecules in the sample contacted with the array to the number ofbinding domains in the array is less than about 1:500.
 46. The method ofclaim 41 wherein sample comprises at least one analyte molecule at afirst concentration and the method further comprises diluting the sampleto create a diluted sample, wherein the diluted sample comprises theanalyte molecules at a second concentration; contacting the array withthe diluted sample such that the ratio of analyte molecules to the totalnumber of binding domains in the array is between 1:1 and 1:500; anddetermining the number of vessels of said array which contain an analytemolecule.
 47. The method of claim 46 wherein the ratio is less thanabout 1:1.
 48. The method of claim 46 wherein the ratio is less thanabout 1:5.
 49. The method of claim 46 wherein the ratio is less thanabout 1:10.
 50. The method of claim 46 wherein the ratio is less thanabout 1:100.
 51. The method of claim 46 wherein the ratio is less thanabout 1:500.
 52. A kit for the measurement of an analyte of interest ina sample, the kit comprising: a. A surface comprising a binding reagentfor the analyte and an anchoring reagent comprising an anchoringsequence complementary to an amplicon sequence; and b. In one or morecontainers, compartments, or vessels: i. Two detection reagents for theanalyte, wherein the two detection reagents comprise a first proximityprobe and a second proximity probe, respectively; ii. one or moreconnector oligonucleotides including a first connector probecomplementary to a first region of the first proximity probe and a firstregion on the second proximity probe, and a second connector probecomplementary to a second non-overlapping region of the first proximityprobe and a second non-overlapping region of the second proximity probe;and iii. One or more detection probes complementary to the detectionprobe sequence.
 53. The kit of claim 52 further comprising (b)(iv) DNApolymerase.
 54. The kit of claim 52 wherein each of the one or moredetection probes comprise a detectable label.
 55. The kit of claim 54wherein the detectable label is an ECL label and the kit furthercomprises (b)(v) an ECL co-reactant.
 56. A kit for the measurement of ananalyte of interest in a sample, the kit comprising: a. A surfacecomprising a binding reagent for the analyte and an anchoring reagentcomprising an anchoring sequence complementary to an amplicon sequence;and b. In one or more containers, compartments, or vessels: i. Twodetection reagents for the analyte, wherein the two detection reagentscomprise a first proximity probe and a second proximity probe,respectively; ii. One or more connector oligonucleotides including afirst circularization probe complementary to a first region of the firstproximity probe and a first region on the second proximity probe, and asecond circularization probe complementary to a second non-overlappingregion of the first proximity probe and a second non-overlapping regionof the second proximity probe; and iii. One or more detection probescomplementary to the detection probe sequence.
 57. The kit of claim 56further comprising (b)(iv) DNA polymerase.
 58. The kit of claim 56wherein each of the one or more detection probes comprise a detectablelabel.
 59. The kit of claim 58 wherein the detectable label is an ECLlabel and the kit further comprises (b)(v) an ECL co-reactant.
 60. A kitfor the measurement of a plurality of analytes of interest in a sample,the kit comprising: a. A surface comprising a plurality of discretebinding domains, each binding domain comprising (i) a binding reagentfor an analyte and (ii) an anchoring reagent comprising an anchoringsequence complementary to an amplicon sequence; and b. In one or morecontainers, compartments, or vessels: i. Two detection reagents for eachanalyte, wherein the two detection reagents comprise a first proximityprobe and a second proximity probe, respectively; iii. one or moreconnector oligonucleotides including a first circularization probecomplementary to a first region of the first proximity probe and a firstregion on the second proximity probe, and a second circularization probecomplementary to a second non-overlapping region of the first proximityprobe and a second non-overlapping region of the second proximity probe;and iii. One or more detection probes complementary to the detectionprobe sequence.
 61. The kit of claim 60 further comprising (b)(iv) DNApolymerase.
 62. The kit of claim 60 wherein each of the one or moredetection probes comprise a detectable label.
 63. The kit of claim 62wherein the detectable label is an ECL label and the kit furthercomprises (b)(v) an ECL co-reactant. 64.-143. (canceled)
 144. The methodof claim 24 wherein the method further comprises exposing the sample toa plurality of binding domains under conditions so that at least oneanalyte molecule is anchored in at least some of the binding domains,wherein each binding domain is positioned within a well of a multi-wellplate and each binding domain defines a binding surface that has abinding reagent immobilized thereon; determining the presence or absenceof an analyte molecule in each binding domain so as to identify thenumber of binding domains that contain an analyte molecule and/or thenumber of binding domains that do not contain an analyte molecule; anddetermining the concentration of analyte molecules in the sample to betested from the number of binding domains that contain and/or do notcontain an analyte molecule.
 145. The method of claim 24 furthercomprising partitioning at least a portion of the analyte molecules inthe sample into a plurality of binding domains, so that, forsubstantially all of the binding domains, each binding domain containseither no analyte molecules or a single analyte molecule; determiningthe presence or absence of an analyte molecule in a plurality of bindingdomains to provide a fraction of the interrogated binding domains thatcontain an analyte molecule; and determining the concentration ofanalyte molecules in the sample from the fraction of interrogatedbinding domains that contain an analyte molecule.
 146. The method ofclaim 24 further comprising partitioning the sample into a plurality ofsecond, smaller samples of equal volume so that at least some of thesecond, smaller samples contain either a single analyte molecule or noanalyte molecules, determining the presence or absence of an analytemolecule in at least a subset of the second samples so as to identifythe fraction of second samples in the subset that contain an analytemolecule; and determining the concentration of analyte molecules in thesample to be tested from the fraction of second samples of the subsetthat contain the analyte molecules.
 147. The method of claim 24 furthercomprising contacting an array with the sample such that the ratio ofthe number of analyte molecules in the sample contacted with the arrayto the number of binding domains in the array is less than 1:1; anddetermining the number of binding domains which contain an analytemolecule, wherein the array comprises a multi-well plate including aplurality of wells, wherein each well comprising a plurality of bindingdomains.